This is a new R21 grant application responsive to PA 02-046, "Innovation Grants for AIDS Research". The overall aim of this study is to determine the phenotype, function, and role of CD4+CD25+ regulatory T cells (T/reg cells) in HIV infected and uninfected humans. Treg cells represent a subset of circulating CD4+CD25+ T cells with suppressive properties. Their significance in vivo was recently shown in a mouse model, where adoptive transfer of T/reg cells prevented intestinal inflammation. There is limited data on the phenotypic characterization of human CD4+CD25+ regulatory T (Trig) cells. In the first specific aim we will sort T/reg cells based upon the expression of CD4 and CD25, and then use additional markers to further refine the phenotype of functional Trig cells using eight-color flow cytometry. The phenotypic characterization will be combined with functional experiments, where the sorted subpopulations of T=g cells will be assayed for their suppressive capacity in vitro. In a cross sectional study, we will quantify the number, frequency, and suppressive activity of T=g cells in PBMC obtained from 20 healthy individuals. In a longitudinal study on 6 donors on a bi-monthly basis we will measure the number, frequency and activity of the T/reg cell population under normal conditions. We will also characterize the mechanism of Treg cell mediated suppression. The second specific aim of this study is to determine the impact of HIV infection on the frequency, absolute numbers and suppressive activity of T=g cells. Our preliminary results demonstrate that Treg cells can suppress HIV specific T cell responses in vitro. We will measure the frequency and absolute numbers of functional T/reg cells in PBMC by eight-color flow cytometry, and assess the suppressive activity of the T/reg cells in vitro by measuring cytokine production and proliferation after stimulation with antigens in cross sectional and longitudinal studies. We hypothesize that Treg cells are induced by HIV infection and that they suppress HIV specific T cell responses early in infection. We will use samples from early primary HIV infection subjects to assess whether Treg cell number and activity is increased. We will also ascertain the impact of highly active antiretroviral therapy (HAART) on T,eg cells, and the effect of treatment interruptions on T/reg cell functions. We also hypothesize that T/reg cells in HIV infected individuals will be infected with HIV, and we will quantitatively assess HIV infection and replication in T/reg cells ompared to other CD4+ T cell subpopulations. The goal of this proposal is to generate data to determine the importance of T=g cells in HIV infection. Manipulation of T=g cells could be critical for vaccine strategies and for therapeutic interventions.